System and method for characterizing drug product impurities

ABSTRACT

Systems and methods for characterizing low molecular weight (LMW) protein drug product impurities are provided. One embodiment uses hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry analysis. After removal of the N-linked glycans from the protein drug product, for example an antibody drug product, the elution of LMW impurities from the HILIC column was determined by the size of the molecular weight species. In some embodiments, the HILIC separation is performed under denaturing conditions, making the detection of LMW forms using this method highly comparable to both SDS-PAGE and CE-SDS methods. LMW drug product impurities include, but are not limited to light chain, half antibody, H2L, H2, HL, HC, peptide backbone-truncated species, and combinations thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority to U.S. ProvisionalPatent Application No. 62/743,632 filed on Oct. 10, 2018, and U.S.Provisional Patent Application No. 62/610,029 filed on Dec. 22, 2017,both of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention is generally directed to protein separation methods andcell culture methods.

BACKGROUND OF THE INVENTION

Monoclonal antibodies (mAbs) have been successfully employed to target awide range of therapeutic areas over the last two decades (Walsh G.,Nature biotechnology, 32:992-1000 (2014); Lawrence S. Naturebiotechnology, 25:380-2 (2007)). While mAbs possess a conserved covalentheterotetrameric structure consisting of two disulfide-linked heavychains, each covalently linked through a disulfide bond to a lightchain, these proteins often contain low levels of product-relatedimpurities even after extensive purification steps. Low molecular weight(LMW) species (e.g. peptide backbone-truncated fragments) and highmolecular weight (HMW) species (e.g. antibody dimer species) are bothexamples of product-related impurities that contribute to the sizeheterogeneity of mAb products. The formation of HMW species within atherapeutic mAb drug product as a result of protein aggregation canpotentially compromise both drug efficacy and safety (e.g. elicitingunwanted immunogenic response) (Rosenberg A S. The AAPS journal,8:E501-7 (2006); Moussa E M, et al. Journal of pharmaceutical sciences,105:417-30(2006)). LMW species of any therapeutic protein may resultfrom host cell protease activity during production. LMW species oftenhave low or substantially reduced activity relative to the monomericform of the antibody, while exposing novel epitopes that can lead toimmunogenicity or potentially impact pharmacokinetic properties in vivo(Vlasak J, Ionescu R. mAbs, 3:253-63 (2011)). As a result, both HMW andLMW species are considered critical quality attributes that areroutinely monitored during drug development and as part of releasetesting of purified drug substance during manufacturing.

Molecular weight heterogeneity of mAb products is traditionallycharacterized by multiple orthogonal analytical methods (Michels D A,Parker M, Salas-Solano O. Electrophoresis, 33:815-26(2012)). One of themost commonly used techniques to assess mAb product purity is SDS-PAGE,performed under non-reducing conditions. During analysis, minor bandscorresponding to LMW species can be routinely observed and quantified,including H2L (2 heavy chains and 1 light chain), H2 (2 heavy chains),HL (1 heavy chain and 1 light chain), HC (1 heavy chain), and LC (1light chain) species, with respect to antibodies (Liu H, Gaza-Bulseco G,Chumsae C, Newby-Kew A. Biotechnology letters, 29:1611-22(2007)).Proteolytic fragments may also be observed. The proposed identity ofeach minor band can be supported by N-terminal sequencing via Edmandegradation, in-gel tryptic digestion followed by mass spectrometryanalysis, and western blot analysis using anti-Fc and anti-light chainantibodies. However, any proposed structures resulting from thesemethods cannot be unambiguously confirmed at the intact protein level.Furthermore, sample preparation conditions employed in SDS-PAGEexperiments can generate LMW artifacts through disulfide bondscrambling, which can lead to overestimations of minor LMW species (ZhuZ C, et al. Journal of pharmaceutical and biomedical analysis,83:89-95(2013)). More recently, capillary electrophoresis-sodium dodecylsulfate (CE-SDS) has emerged as a modern equivalent of SDS-PAGE,offering superior reproducibility, sensitivity, and throughput (RustandiR R, Washabaugh M W, Wang Y. Electrophoresis, 29:3612-20 (2008); LacherN A, et al. Journal of separation science, 33:218-27 (2010); Hunt G,Moorhouse K G, Chen A B. Journal of chromatography A,744:295-301(1996)). During CE-SDS analysis of mAb products, minor peakswith shorter migration times (LMW forms) than the intact antibody can beroutinely observed. Unlike SDS-PAGE analysis, these LMW impuritiescannot be extracted or subjected to further analyses. As a result, theidentities of LMW impurities observed in CE-SDS methods are oftenproposed solely based on empirical knowledge. Thus, an orthogonal methodto directly separate and unambiguously identify the LMW impurities inmAb products is essential for ensuring control of the manufacturingprocess during antibody product development.

Accurate mass measurement of intact mAb proteins by modern massspectrometers has become increasingly popular in the biopharmaceuticalindustry as one of the most reliable identification techniques(Kaltashov I A, et al., Journal of the American Society for MassSpectrometry, 21:323-37 (2010)); Zhang H, Cui W, Gross M L. FEBSletters, 588:308-17(2014)). Specifically, a variety of “hyphenatedchromatography-mass spectrometry” methods have demonstrated thecapability of detecting low-abundance impurities in mAb products andproviding highly detailed analyses that cannot be achieved by eitherSDS-PAGE or CE-SDS methods (Le J C, Bondarenko P V. Journal of theAmerican Society for Mass Spectrometry, 16:307-11 (2005); Haberger M, etal. mAbs, 8:331-9 (2016)). For example, reversed-phase chromatography(RPLC) coupled to mass spectrometry can be used to detect free lightchain and associated post-translational modifications (e.g.cysteinylation and glutathionylation) present in mAb drug products.However, compared to SDS-PAGE and CE-SDS methods, RPLC often lackssufficient resolution to separate LMW species and thus fails toelucidate the complete LMW profile. For example, the identification ofH2L species in mAb drug products has never been reported by RPLC-basedintact mass analysis, owing to its low abundance and poor resolutionfrom the main intact antibody. Another MS-based technique that ispromising for characterizing mAb product-related impurities is nativeelectrospray ionization mass spectrometry (Native ESI-MS), which isparticularly informative when coupled with size exclusion chromatography(SEC) (Haberger M, et al. mAbs, 8:331-9 (2016)). However, the LMWspecies identified in native SEC-MS analysis are often not the same asthose identified by SDS-PAGE or CE-SDS, due to significantly differentexperimental conditions used between methods. Specifically, the samplepreparation required for SDS-PAGE and CE-SDS often starts with proteindenaturation, where the non-covalent interactions between the N-terminalregions of HC-LC pairs and the C-terminal regions of the HC-HC pairs aredisrupted. As a result, LMW impurities such as H2L, half antibody, andfree light chain species are able to dissociate from the mAb molecule ifthe interchain disulfide bonds are broken. In comparison, native SEC-MSanalyzes the mAb samples under near native conditions, permitting thestrong non-covalent interchain interactions to be preserved and allowingthe four-chain structure of the mAb molecule to be maintained even ifthe interchain disulfide bonds are broken. Although advances in SECcolumn chemistry have made it possible to use denaturing buffers (e.g.30% acetonitrile, 0.1% FA and 0.1% TFA) that are normally used inreversed-phase chromatography for SEC separation and direct coupling toonline mass spectrometry analysis (Liu H, Gaza-Bulseco G, Chumsae C.Journal of the American Society for Mass Spectrometry,20:2258-64(2009)), the LC resolution is still sub-optimal to detect manyLMW species.

Thus, it is an object of the invention to provide systems and methodsfor the characterization of LMW protein drug impurities.

It is another object of the invention to provide protein drug productswith reduced levels of impurities.

It is still another object of the invention to provide methods ofproducing protein drug products with reduced protein drug productimpurities.

SUMMARY OF THE INVENTION

Systems and methods for characterizing low molecular weight (LMW)protein drug product impurities are provided. One embodiment useshydrophilic interaction chromatography (HILIC) coupled to massspectrometry analysis. After removal of the N-linked glycans from theprotein drug product, for example an antibody drug product, the elutionof LMW impurities from the HILIC column is determined by the size of themolecular weight species. In some embodiments, the HILIC separation isperformed under denaturing conditions, making the detection of LMW formsusing this method highly comparable to both SDS-PAGE and CE-SDS methods.LMW drug product impurities include, but are not limited to light chain,half antibody, H2L, H2, HL, HC, peptide backbone-truncated species, andcombinations thereof.

The disclosed HILIC-MS systems and methods provide detailed LMWidentification information. The reliable identification and detailedstructural information revealed by HILIC-MS analysis is highly valuablefor in-depth characterization of LMW impurities in protein drugproducts, which is often required for late-stage molecule development.Furthermore, because the disclosed HILIC-MS system and methods usegentler sample preparations than either SDS-PAGE or CE-SDS does, it isless likely to generate LMW artifacts. The HILIC-MS systems and methodscan be used as a semi-quantitative analysis to compare the LMW impurityprofile between samples or simply applied qualitatively.

One embodiment provides a protein drug product containing a protein drugand an excipient, wherein the protein drug product comprises betweenabout 0.05 and about 30.0% w/w of low molecular weight protein drugimpurities. The protein drug product can be an antibody, a fusionprotein, recombinant protein, or a combination thereof In otherembodiments, the drug product contains between about 0.05% to about 25%,or about 0.05% to about 15%, or about 0.05% to about 10%, or about 0.05%to about 5%, or about 1 to about 25%, about 1 to about 15%, about 1 toabout 10%, or about 1 to about 5% w/w of low molecular weight proteindrug impurities.

Another embodiment provides a method for characterizing low molecularweight protein drug product impurities including the steps of

-   -   i) deglycosylating a protein drug product sample,    -   ii) separating protein components of the protein drug product        sample by hydrophilic interaction chromatography, and    -   iii) analyzing the separated protein components by mass        spectroscopy to characterize low molecular weight protein drug        product impurities in the protein drug product sample.        The method further provides an optional reducing step. The        reducing step may take place in between step i) and step ii).        The protein drug product sample can be taken from a fed-batch        culture. As noted above, the protein drug product can be an        antibody, a fusion protein, recombinant protein, or a        combination thereof.

Still another embodiment provides a method of producing an antibody,including the steps of culturing cells producing the antibody in a cellculture, obtaining a sample from the cell culture, characterizing andquantifying low molecular weight impurities in the sample according tothe method described above and modifying one or more culture conditionsof the cell culture to reduce the amount of characterized low molecularprotein drug impurities produced during cell culture of the antibody. Insome embodiments, the sample is taken during the cell culture at anyinterval. In other embodiments, the sample is taken following productionculture, following protein harvest or following purification. The one ormore conditions of the cell culture that are changed to reduce theamount of low molecular weight protein drug impurities can be selectedfrom the group consisting of temperature, pH, cell density, amino acidconcentration, osmolality, growth factor concentration, agitation, gaspartial pressure, surfactants, or combinations thereof. The cells can beeukaryotic or prokaryotic. The cells can be Chinese Hamster Ovary (CHO)cells (e.g. CHO K1, DXB-11 CHO, Veggie-CHO), COS cells (e.g. COS-7),retinal cells, Vero cells, CV1 cells, kidney cells (e.g. HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK21), HeLa cells, HepG2 cells, WI38 cells,MRC 5 cells, Colo25 cells, HB 8065 cells, HL-60 cells, lymphocyte cells,e.g. autologous T cells, Jurkat (T lymphocytes) or Daudi (Blymphocytes), A431 (epidermal) cells, U937 cells, 3T3 cells, L cells,C127 cells, SP2/0 cells, NS-0 cells, MMT cells, stem cells, tumor cells,and a cell line derived from any of the aforementioned cells. In oneembodiment the cells are hybridoma or quadroma cells. Still anotherembodiment provides an antibody produced by the methods describedherein.

Yet another embodiment provides a system for characterizing lowmolecular weight drug impurities. The system includes a hydrophilicinteraction liquid chromatography system including a hydrophilicinteraction liquid chromatography (HILIC) column linked to mobile phaseA and mobile phase B as exemplified herein, and the HILIC column is influid communication with a mass spectroscopy system.

In yet another embodiment, the invention relates to use of the methodaccording to the invention for determination of stability and forceddegradation studies of a protein drug product.

In a further embodiment, the invention relates to use of a systemaccording to the invention for determination of stability and forceddegradation studies of a protein drug product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are chromatograms of HILIC separation of mAb-1 drugsubstance sample. FIG. 1A is the ultraviolet profile and FIG. 1B is theHILIC profile. The UV signal was amplified 10 times in FIG. 1A to bettervisualize the LMW impurities.

FIG. 2A shows the extracted ion chromatograms (XICs) of different lightchain variants using the m/z of the most abundant charge state. FIG. 2Bshows the deconvoluted mass spectrum of light chain with β-elimination.FIG. 2C shows the deconvoluted mass spectrum of light chain modified by2-mercaptoacetic acid, unmodified light chain and light chain withC-terminal residues clipped. FIG. 2D shows the deconvoluted massspectrum of light chain with cysteinylation. FIG. 2E shows thedeconvoluted mass spectrum of light chain with glutathionylation.

FIG. 3A shows the deconvoluted mass spectrum of Fab fragments. FIG. 3Bshows the deconvoluted mass spectrum of half antibody. FIG. 3C shows thedeconvoluted mass spectrum of Fab-truncated fragments. FIG. 3D shows thedeconvoluted mass spectrum of H2.

FIG. 4 shows The HILIC-UV analysis of deglycosylated mAb-1 sampletreated by DTT (arrow #1) and L-cysteine (arrow #2). Arrow #3 points tothe trace with untreated mAb-1. The signal of untreated sample andL-cysteine treated sample were amplified 10 times and 2 times,respectively, to better visualize the LMW impurities.

FIG. 5A shows light chain species identified in HILIC-MS analysis ofmAb-1 after treated with DTT. FIG. 5B shows heavy chain speciesidentified in HILIC-MS analysis of mAb-1 after treated with DTT. FIG. 5Cshows half antibody species identified in HILIC-MS analysis of mAb-1after treated with DTT. FIG. 5D shows heavy chain dimer speciesidentified in HILIC-MS analysis of mAb-1 after treated with DTT. FIG. 5Eshows H2L species identified in HILIC-MS analysis of mAb-1 after treatedwith DTT. FIG. 5F shows full antibody species identified in HILIC-MSanalysis of mAb-1 after treated with DTT.

FIG. 6A shows an MS2 spectrum of light chain C-terminal peptides fromLys-C digestion under non-reduced conditions wherein the C-terminal Cysis modified by iodoacetamide. FIG. 6B shows an MS2 spectrum of lightchain C-terminal peptides from Lys-C digestion under non-reducedconditions wherein the C-terminal Cys is modified by unknownmodification of +89.98 Da.

FIG. 7A shows the extracted ion chromatogram (XIC) of light chainC-terminal peptide with unknown modification of +89.98 Da non-reducedLys-C digests. FIG. 7B shows the extracted ion chromatogram (XIC) oflight chain C-terminal peptide with unknown modification of +89.98 Dafrom reduction of the non-reduced Lys-C digests.

FIG. 8 is the proposed structure (2-mercaptoacetic acid) of the unknownmodification.

FIG. 9 is an exemplary HILIC-MS system.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The use of the terms “a,” “an,” “the,” and similar referents in thecontext of describing the presently claimed invention (especially in thecontext of the claims) are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

Use of the term “about” is intended to describe values either above orbelow the stated value in a range of approx. +/−10%; in otherembodiments the values may range in value either above or below thestated value in a range of approx. +/−5%; in other embodiments thevalues may range in value either above or below the stated value in arange of approx. +/−2%; in other embodiments the values may range invalue either above or below the stated value in a range of approx.+/−1%. The preceding ranges are intended to be made clear by context,and no further limitation is implied. All methods described herein canbe performed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

The term “low molecular weight (LMW) protein drug impurity” includes butis not limited to precursors, degradation products, truncated species,proteolytic fragments including Fab fragments, Fc or heavy chainfragments, ligand or receptor fragments, H2L (2 heavy chains and 1 lightchain), H2 (2 heavy chains), HL (1 heavy chain and 1 light chain), HC (1heavy chain), and LC (1 light chain) species. A LMW protein drugimpurity can be any variant which is an incomplete version of theprotein product, such as one or more components of a multimeric protein.Protein drug impurity, drug impurity or product impurity are terms thatmay be used interchangeably throughout the specification and include LMWprotein drug impurities. LMW drug or product impurities are generallyconsidered molecular variants with properties such as activity,efficacy, and safety that may be different from those of the desireddrug product.

Degradation of protein product is problematic during production of theprotein drug product in cell culture systems. For example, proteolysisof a protein product may occur due to release of proteases in cellculture medium. Medium additives, such as soluble iron sources added toinhibit metalloproteases, or serine and cysteine proteases inhibitors,have been implemented in cell culture to prevent degradation (Clincke,M.-F., et al, BMC Proc. 2011, 5, P 115). C-terminal fragments may becleaved during production due to carboxyl peptidases in the cell culture(Dick, L W et al, Biotechnol Bioeng 2008; 100:1132-43). Subsequently,there is a need to profile and monitor LMW impurities.

“Protein” refers to a molecule comprising two or more amino acidresidues joined to each other by a peptide bond. Protein includespolypeptides and peptides and may also include modifications such asglycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, alkylation, hydroxylation and ADP-ribosylation.Proteins can be of scientific or commercial interest, includingprotein-based drugs, and proteins include, among other things, enzymes,ligands, receptors, antibodies and chimeric or fusion proteins. Proteinsare produced by various types of recombinant cells using well-known cellculture methods, and are generally introduced into the cell by geneticengineering techniques (e.g., such as a sequence encoding a chimericprotein, or a codon-optimized sequence, an intronless sequence, etc.)where it may reside as an episome or be intergrated into the genome ofthe cell.

“Antibody” refers to an immunoglobulin molecule consisting of fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain has a heavy chainvariable region (HCVR or VH) and a heavy chain constant region. Theheavy chain constant region contains three domains, CH1, CH2 and CH3.Each light chain has a light chain variable region and a light chainconstant region. The light chain constant region consists of one domain(CL). The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term “antibody” includesreference to both glycosylated and non-glycosylated immunoglobulins ofany isotype or subclass. The term “antibody” includes antibody moleculesprepared, expressed, created or isolated by recombinant means, such asantibodies isolated from a host cell transfected to express theantibody. The term antibody also includes bispecific antibody, whichincludes a heterotetrameric immunoglobulin that can bind to more thanone different epitope. Bispecific antibodies are generally described inUS Patent Application Publication No. 2010/0331527, which isincorporated by reference into this application.

“Fc fusion proteins” comprise part or all of two or more proteins, oneof which is an Fc portion of an immunoglobulin molecule, which are nototherwise found together in nature. Preparation of fusion proteinscomprising certain heterologous polypeptides fused to various portionsof antibody-derived polypeptides (including the Fc domain) has beendescribed, e.g., by Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al.,“Construction of Immunoglobulin Fusion Proteins”, in Current Protocolsin Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992. “Receptor Fcfusion proteins” comprise one or more extracellular domain(s) of areceptor coupled to an Fc moiety, which in some embodiments comprises ahinge region followed by a CH2 and CH3 domain of an immunoglobulin. Insome embodiments, the Fc-fusion protein comprises two or more distinctreceptor chains that bind to a one or more ligand(s). For example, anFc-fusion protein is a trap, such as for example an IL-1 trap or VEGFtrap.

“Cell culture” refers to the propagation or proliferation of cells in avessel, such as a flask or bioreactor, and includes but is not limitedto fed-batch culture, continuous culture, perfusion culture and thelike.

The term “HILIC or HILIC chromatography” refers to hydrophilicinteraction liquid chromatography or hydrophilic interactionchromatography, and is considered a well-known term of the art.

Proteins of Interest

Any protein of interest suitable for expression in prokaryotic oreukaryotic cells can be used in the engineered host cell systemsprovided. For example, the protein of interest includes, but is notlimited to, an antibody or antigen-binding fragment thereof, a chimericantibody or antigen-binding fragment thereof, an ScFv or fragmentthereof, an Fc-fusion protein or fragment thereof, a growth factor or afragment thereof, a cytokine or a fragment thereof, or an extracellulardomain of a cell surface receptor or a fragment thereof. Proteins ofinterest may be simple polypeptides consisting of a single subunit, orcomplex multisubunit proteins comprising two or more subunits. Theprotein of interest may be a biopharmaceutical product, food additive orpreservative, or any protein product subject to purification and qualitystandards.

In some embodiments, the protein product (protein of interest) is anantibody, a human antibody, a humanized antibody, a chimeric antibody, amonoclonal antibody, a multispecific antibody, a bispecific antibody, anantigen binding antibody fragment, a single chain antibody, a diabody,triabody or tetrabody, a Fab fragment or a F(ab′)2 fragment, an IgDantibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. Inone embodiment, the antibody is an IgG1 antibody. In one embodiment, theantibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4antibody. In one embodiment, the antibody is a chimeric IgG2/IgG4antibody. In one embodiment, the antibody is a chimeric IgG2/IgG1antibody. In one embodiment, the antibody is a chimeric IgG2/IgG1/IgG4antibody.

In some embodiments, the antibody is selected from the group consistingof an anti-Programmed Cell Death 1 antibody (e.g. an anti-PD1 antibodyas described in U.S. Pat. Appln. Pub. No. US2015/0203579A1), ananti-Programmed Cell Death Ligand-1 (e.g. an anti-PD-L1 antibody asdescribed in in U.S. Pat. Appln. Pub. No. US2015/0203580A1), ananti-Dll4 antibody, an anti-Angiopoetin-2 antibody (e.g. an anti-ANG2antibody as described in U.S. Pat. No. 9,402,898), ananti-Angiopoetin-Like 3 antibody (e.g. an anti-AngPtl3 antibody asdescribed in U.S. Pat. No. 9,018,356), an anti-platelet derived growthfactor receptor antibody (e.g. an anti-PDGFR antibody as described inU.S. Pat. No. 9,265,827), an anti-Erb3 antibody, an anti-ProlactinReceptor antibody (e.g. anti-PRLR antibody as described in U.S. Pat. No.9,302,015), an anti-Complement 5 antibody (e.g. an anti-C5 antibody asdescribed in U.S. Pat. Appln. Pub. No US2015/0313194A1), an anti-TNFantibody, an anti-epidermal growth factor receptor antibody (e.g. ananti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or ananti-EGFRvIII antibody as described in U.S. Pat. Appln. Pub. No.US2015/0259423A1), an anti-Proprotein Convertase Subtilisin Kexin-9antibody (e.g. an anti-PCSK9 antibody as described in U.S. Pat. No.8,062,640 or U.S. Pat. Appln. Pub. No. US2014/0044730A1), an anti-GrowthAnd Differentiation Factor-8 antibody (e.g. an anti-GDF8 antibody, alsoknown as anti-myostatin antibody, as described in U.S. Pat Nos.8,871,209 or 9,260,515), an anti-Glucagon Receptor (e.g. anti-GCGRantibody as described in U.S. Pat. Appln. Pub. Nos. US2015/0337045A1 orUS2016/0075778A1), an anti-VEGF antibody, an anti-IL1R antibody, aninterleukin 4 receptor antibody (e.g. an anti-IL4R antibody as describedin U.S. Pat. Appln. Pub. No. US2014/0271681A1 or U.S. Pat Nos. 8,735,095or 8,945,559), an anti-interleukin 6 receptor antibody (e.g. ananti-IL6R antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617or 9,173,880), an anti-IL1 antibody, an anti-IL2 antibody, an anti-IL3antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6antibody, an anti-IL7 antibody, an anti-interleukin 33 (e.g. anti-IL33antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0271658A1 orUS2014/0271642A1), an anti-Respiratory syncytial virus antibody (e.g.anti-RSV antibody as described in U.S. Pat. Appln. Pub. No.US2014/0271653A1), an anti-Cluster of differentiation 3 (e.g. ananti-CD3 antibody, as described in U.S. Pat. Appln. Pub. Nos.US2014/0088295A1 and US20150266966A1, and in U.S. Application No.62/222,605), an anti-Cluster of differentiation 20 (e.g. an anti-CD20antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 andUS20150266966A1, and in U.S. Pat. No. 7,879,984), an anti-CD19 antibody,an anti-CD28 antibody, an anti-Cluster of Differentiation-48 (e.g.anti-CD48 antibody as described in U.S. Pat. No. 9,228,014), an anti-Feld1 antibody (e.g. as described in U.S. Pat. No. 9,079,948), ananti-Middle East Respiratory Syndrome virus (e.g. an anti-MERS antibodyas described in U.S. Pat. Appln. Pub. No. US2015/0337029A1), ananti-Ebola virus antibody (e.g. as described in U.S. Pat. Appln. Pub.No. US2016/0215040), an anti-Zika virus antibody, an anti-LymphocyteActivation Gene 3 antibody (e.g. an anti-LAG3 antibody, or an anti-CD223antibody), an anti-Nerve Growth Factor antibody (e.g. an anti-NGFantibody as described in U.S. Pat. Appln. Pub. No. US2016/0017029 andU.S. Pat. Nos. 8,309,088 and 9,353,176) and an anti-Activin A antibody.In some embodiments, the bispecific antibody is selected from the groupconsisting of an anti-CD3×anti-CD20 bispecific antibody (as described inU.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US20150266966A1), ananti-CD3×anti-Mucin 16 bispecific antibody (e.g., an anti-CD3×anti-Muc16bispecific antibody), and an anti-CD3×anti-Prostate-specific membraneantigen bispecific antibody (e.g., an anti-CD3×anti-PSMA bispecificantibody). In some embodiments, the protein of interest is selected fromthe group consisting of abciximab, adalimumab, adalimumab-atto,ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab,basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab,blinatumomab, brentuximab vedotin, brodalumab, canakinumab, capromabpendetide, certolizumab pegol, cemiplimab, cetuximab, denosumab,dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab,emicizumab-kxwh, emtansinealirocumab, evinacumab, evolocumab, fasinumab,golimumab, guselkumab, ibritumomab tiuxetan, idarucizumab, infliximab,infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab,necitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab,ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab,pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab,reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab,tocilizumab, tocilizumab, trastuzumab, trevogrumab, ustekinumab, andvedolizumab.

In some embodiments, the protein of interest is a recombinant proteinthat contains an Fc moiety and another domain, (e.g., an Fc-fusionprotein). In some embodiments, an Fc-fusion protein is a receptorFc-fusion protein, which contains one or more extracellular domain(s) ofa receptor coupled to an Fc moiety. In some embodiments, the Fc moietycomprises a hinge region followed by a CH2 and CH3 domain of an IgG. Insome embodiments, the receptor Fc-fusion protein contains two or moredistinct receptor chains that bind to either a single ligand or multipleligands. For example, an Fc-fusion protein is a TRAP protein, such asfor example an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcPligand binding region fused to the Il-1R1 extracellular region fused toFc of hIgG1; see U.S. Pat. No. 6,927,004, which is herein incorporatedby reference in its entirety), or a VEGF trap (e.g., aflibercept orziv-aflibercept, which comprises the Ig domain 2 of the VEGF receptorFlt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc ofhIgG1; see U.S. Pat. Nos. 7,087,411 and 7,279,159). In otherembodiments, an Fc-fusion protein is a ScFv-Fc-fusion protein, whichcontains one or more of one or more antigen-binding domain(s), such as avariable heavy chain fragment and a variable light chain fragment, of anantibody coupled to an Fc moiety.

Cell Culture

In protein production, a “fed-batch cell culture” or “fed-batch culture”refers to a batch culture wherein the cells and culture medium aresupplied to the culturing vessel initially, and additional culturenutrients are slowly fed, in discrete increments, to the culture duringculturing, with or without periodic cell and/or product harvest beforetermination of culture. Fed-batch culture includes “semi-continuousfed-batch culture” wherein periodically whole culture (which may includecells and medium) is removed and replaced by fresh medium. Fed-batchculture is distinguished from simple “batch culture” whereas allcomponents for cell culturing (including the animal cells and allculture nutrients) are supplied to the culturing vessel at the start ofthe culturing process in batch culture. Fed-batch culture may bedifferent from “perfusion culture” insofar as the supernatant is notremoved from the culturing vessel during a standard fed-batch process,whereas in perfusion culturing, the cells are restrained in the cultureby, e.g., filtration, and the culture medium is continuously orintermittently introduced and removed from the culturing vessel.However, removal of samples for testing purposes during fed-batch cellculture is contemplated. The fed-batch process continues until it isdetermined that maximum working volume and/or protein production isreached, and protein is subsequently harvested.

The phrase “continuous cell culture” relates to a technique used to growcells continually, usually in a particular growth phase. For example, ifa constant supply of cells is required, or the production of aparticular protein of interest is required, the cell culture may requiremaintenance in a particular phase of growth. Thus, the conditions mustbe continually monitored and adjusted accordingly in order to maintainthe cells in that particular phase.

The terms “cell culture medium” and “culture medium” refer to a nutrientsolution used for growing mammalian cells that typically provides thenecessary nutrients to enhance growth of the cells, such as acarbohydrate energy source, essential (e.g. phenylalanine, valine,threonine, tryptophan, methionine, leucine, isoleucine, lysine, andhistidine) and nonessential (e.g. alanine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, proline, serine, andtyrosine) amino acids, trace elements, energy sources, lipids, vitamins,etc. Cell culture medium may contain extracts, e.g. serum or peptones(hydrolysates), which supply raw materials that support cell growth.Media may contain yeast-derived or soy extracts, instead ofanimal-derived extracts. Chemically defined medium refers to a cellculture medium in which all of the chemical components are known (i.e.have a known chemical structure). Chemically defined medium is entirelyfree of animal-derived components, such as serum- or animal-derivedpeptones. In one embodiment, the medium is a chemically defined medium.

The solution may also contain components that enhance growth and/orsurvival above the minimal rate, including hormones and growth factors.The solution may be formulated to a pH and salt concentration optimalfor survival and proliferation of the particular cell being cultured.

A “cell line” refers to a cell or cells that are derived from aparticular lineage through serial passaging or subculturing of cells.The term “cells” is used interchangeably with “cell population”.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes, such as bacterial cells, mammalian cells, human cells,non-human animal cells, avian cells, insect cells, yeast cells, or cellfusions such as, for example, hybridomas or quadromas. In certainembodiments, the cell is a human, monkey, ape, hamster, rat or mousecell. In other embodiments, the cell is selected from the followingcells: Chinese Hamster Ovary (CHO) (e.g. CHO K1, DXB-11 CHO,Veggie-CHO), COS (e.g. COS-7), retinal cell, Vero, CV1, kidney (e.g.HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5,Colo25, HB 8065, HL-60, lymphocyte, e.g. Jurkat (T lymphocyte) or Daudi(B lymphocyte), A431 (epidermal), U937, 3T3, L cell, C127 cell, SP2/0,NS-0, MMT cell, stem cell, tumor cell, and a cell line derived from anaforementioned cell. In some embodiments, the cell comprises one or moreviral genes, e.g. a retinal cell that expresses a viral gene (e.g. aPER.C6® cell). In some embodiments, the cell is a CHO cell. In otherembodiments, the cell is a CHO K1 cell.

II. Systems for Characterizing Low Molecular Weight Protein DrugImpurities

Multisubunit therapeutic proteins, particularly monoclonal antibody(mAb)-based therapeutics are inherently heterogeneous with respect tosize due to their complex multi-chain structure and the propensity toaccommodate multiple enzymatic and chemical post-translationalmodifications. Although the levels of size variants within a mAb drugproduct can be readily quantitated by a variety of biophysical methods,unambiguous identification of those product-related impurities has beenparticularly challenging.

Hydrophilic interaction chromatography (HILIC) coupled with massspectrometry analysis was used to identify low levels of lower molecularweight (LMW) impurities present within a purified drug substance lot.After removal of N-linked glycans, the HILIC method separatesmAb-related LMW impurities with a size-based elution order. Thesubsequent mass measurement from a high-resolution accurate massspectrometer provides direct and unambiguous identification of a varietyof low-abundance LMW impurities in a single LC-MS analysis. Free lightchain, half antibody, H2L species (antibody possessing a single lightchain) and peptide backbone-truncated species can all be confidentlyidentified and elucidated in great detail, including the truncationsites and associated post-translational modifications. Such detailedinformation that cannot be acquired by traditional purity assays (e.g.SDS-PAGE and CE-SDS) is of great importance to elucidate how the LMWimpurities are formed, making this new method a valuable addition toanalytical characterization portfolio. This is the first known reportwhere the H2L species was directly detected in a mAb drug substancesample by intact mass analysis without prior enrichment.

A. Systems for Characterizing LMW Impurities

The system includes a hydrophilic interaction liquid chromatographysystem including a hydrophilic interaction liquid chromatography (HILIC)column linked to mobile phase A and mobile phase B, and the HILIC columnis in fluid communication with a mass spectroscopy system. The HILICcolumn is suitable for use with deglycosylated proteins. In oneembodiment, the disclosed system contains a Waters ACQUITY™ UPLCGlycoprotein Amide column (300 Å, 1.7 μm, 2.1×150 mm) with a WatersACQUITY™ UPLC system. The column is typically operated at 60° C.Exemplary mobile phases include 0.1% TFA in water as mobile phase A,0.1% TFA in acetonitrile as mobile phase B, and the flow rate was set to0.2 mL/min. The UV traces are typically recorded at 215 and 280 nm. Inone embodiment, the separation is achieved over 55 min with an initial0.5 min hold at 15% A, followed by an increase to 25% A over the next0.5 min, and another linear increase to 40% A over the next 40 minutes.The gradient can be ramped to 100% A over 1 min and held for 2 minutes,before dropping to 15% A in 1 min, and then maintained at initialcondition for over 10 minutes to equilibrate the column for the nextrun.

The UPLC is directly coupled to a mass spectrometer, for example aThermo Scientific Q Exactive hybrid quadrupole Orbitrap massspectrometer. The capillary voltage is typically set at 4.0 kV, with asheath gas flow rate of 40 arbitrary units and auxiliary gas flow rateof 15 arbitrary units. The capillary temperature is generally set at350° C. and the probe heater temperature is generally set at 400° C.Mass spectra are acquired in the mass range of m/z 800-4000. In oneembodiment, the raw data is deconvoluted using Intact Mass™ softwaredeveloped by Protein Metrics.

B. Methods of Characterizing LMW Impurities

The disclosed systems and methods can be used to characterize LMWprotein drug impurities. One embodiment provides a method forcharacterizing low molecular weight protein drug product impuritiesincluding the steps of deglycosylating a protein drug product sample,separating protein components of the protein drug product sample byhydrophilic interaction chromatography, and analyzing the separatedprotein components by mass spectroscopy to characterize low molecularweight protein drug product impurities in the protein drug productsample. In one embodiment the protein drug product sample is taken fromor purified from a fed-batch cell culture, a continuous cell culture ora perfusion cell culture. Exemplary protein drug product includes butare not limited to an antibody, a fusion protein, recombinant protein,or a combination thereof. Exemplary low molecular weight protein drugproduct impurities include but are not limited to precursors,degradation products, truncated species, proteolytic fragments includingFab, ligand or receptor fragments or heavy chain fragments, free lightchain, half antibody, H2L, H2, HL, HC, or a combination thereof.

Another embodiment includes a step of reducing the sample. Exemplaryreducing agents include but are not limited to dithiothreitol (DTT, CAS3483-12-3), beta-mercaptoethanol (BME, 2BME, 2-ME, b-mer, CAS 60-24-2),2-aminoethanethiol (2-MEA-HCl, also called cysteamine-HCl, CAS156-57-0), Tris (2-carboxyethyl) phosphine hydrochloride, (TCEP, CAS5961-85-3), cysteine hydrochloride (Cys-HCl, CAS 52-89-1), or2-mercaptoethanesulfonic acid sodium salt (MESNA). Other methods forreducing protein bonds are known in the art, such as an immobilizedreductant column which contains resin to which a thiol-based reducingagent has been immobilized to enable the solid-phase reduction ofpeptide and protein disulfide bonds. Reducing agents, includingoxidizing agents, which are suitable for reducing chemical interactionbetween polypeptides are also envisioned.

Exemplary elution profiles are discussed above.

C. Methods of Producing High Purity Protein Drug Products

One embodiment provides a method of producing an antibody including thesteps of culturing cells producing the antibody, for example in afed-batch culture, obtaining a sample from the cell culture,characterizing and quantifying low molecular weight impurities in thesample using the systems and methods disclosed herein and modifying oneor more culture conditions of the cell culture to reduce the amount ofcharacterized low molecular protein drug impurities produced during cellculture of the antibody. Typically, the conditions are changed to havethe protein drug impurities in a range of 0.05% and 30.0%, preferably0.05% to 15%, 0.05% to 10%, 0.05% to 5%, or 0.05% to 2% (w/w).

The one or more conditions of the cell culture that are changed toreduce the amount of low molecular weight protein drug impurities areselected from the group consisting of temperature, pH, cell density,amino acid concentration, osmolality, growth factor concentration,agitation, gas partial pressure, surfactants, or combinations thereof.

In one embodiment the cells producing the antibody are Chinese hamsterovary cells. In other embodiments, the cells are hybridoma cells.

Another embodiment provides an antibody produced according the methodsprovided herein have 1 to 5%, 5 to 10%, 10 to 15%, 15 to 20% proteindrug impurities.

EXAMPLES Example 1 HILIC Separation of mAb-1 Drug Substance SampleMaterials

For this study, a recombinant IgG1 mAb (mAb-1) made by Regeneron wasused. Peptide-N-Glycosidase F (PNGase F, #P0704L) was purchased from NewEngland Biolabs, 1 M Tris-hydrochloride pH 7.5 solution (#15567-027) waspurchased from Invitrogen, dithiothreitol (DTT, #20291) was purchasedfrom Thermo Fisher Scientific, and L-cysteine (#168149-25G) waspurchased from Sigma-Aldrich. Acetonitrile (LC-MS grade, #A955-4) andtrifluoroacetic acid (TFA, #PI28904) were purchased from FisherScientific. Milli-Q water was provided in-house.

Methods

Deglycosylation of mAb-1 and Limited Reduction by DTT and L-Cysteine

The mAb-1 sample was diluted to a final concentration of 5 μg/μL using100 mM Tri-HCl (pH 7.5). PNGase F was added at an enzyme to substrateratio of 1 unit/10 μg protein. The deglycosylation reaction wasconducted at 37° C. for 3 hours. To initiate the limited reduction byDTT, a 20 μg aliquot of the deglycosylated mAb-1 sample was reduced with5 mM DTT before immediate injection onto the HILIC column for online UVand mass spectrometry analysis. To initiate the limited reduction byL-cysteine, a 20 μg aliquot of the deglycosylated mAb-1 sample wasreduced with 5 mM L-cysteine and then at 37° C. for 30 minutes beforeinjection onto the HILIC column for online UV and mass spectrometryanalysis.

HILIC-UV and HILIC-MS Analysis

A Waters ACQUITY UPLC Glycoprotein Amide column (300 Å, 1.7 μm, 2.1×150mm) was used on a Waters ACQUITY UPLC system for all HILIC separations.The column was operated at 60° C. The mobile phases were 0.1% TFA inwater as mobile phase A, 0.1% TFA in acetonitrile as mobile phase B, andthe flow rate was set to 0.2 mL/min. The UV traces were recorded at 215and 280 nm. The separation was achieved over 55 min with an initial 0.5min hold at 15% A, followed by an increase to 25% A over the next 0.5min, and another linear increase to 40% A over the next 40 minutes. Thegradient was then ramped to 100% A over 1 min and held for 2 minutes,before dropping to 15% A in 1 min, and then maintained at initialcondition for over 10 minutes to equilibrate the column for the nextrun. The UPLC was directly coupled to a Thermo Scientific Q Exactivehybrid quadrupole Orbitrap mass spectrometer. The capillary voltage wasset at 4.0 kV, with a sheath gas flow rate of 40 arbitrary units andauxiliary gas flow rate of 15 arbitrary units. The capillary temperaturewas set at 350° C. and the probe heater temperature was set at 400° C.Mass spectra were acquired in the mass range of m/z 800-4000. The rawdata were deconvoluted using Intact Mass™ software developed by ProteinMetrics.

Results

A recombinant IgG1 mAb (mAb-1) drug substance sample was used as a modelmolecule. After treatment with PNGase F to remove N-linked glycans oneach heavy chain, the deglycosylated mAb-1 sample was separated on aHILIC column and analyzed by both photodiode array (PDA) detection (atboth 280 nm and 215 nm) and mass spectrometry analysis (FIGS. 1A and1B). As shown in the UV profile (FIG. A1, bottom trace), the overalllevel of LMW impurities in mAb-1 drug substance sample was very low,suggesting a successful purification process. By comparing the total ionchromatogram (TIC) generated from mass spectrometry analysis to the UVchromatogram, an additional peak (with a retention time at ˜18.2 min)was observed in the TIC trace, which corresponded to the major glycanform (GOF) released from mAb-1 by PNGase F. As oligosaccharides do notexhibit UV absorption at either 280 nm or 215 nm, they are invisible inUV detection. In addition to the PNGase F reagent peak, all other minorpeaks were identified as mAb-1 related LMW impurities, specifically,free light chain (w/ multiple modifications), Fab fragments,half-antibody, Fab-truncated species and H2L species (FIGS. 1A and 1B).The elution order of these LMW impurities on the HILIC column wascorrelated with their relative size, with smaller fragments elutingearlier than the larger fragments. It is important to note that removingthe N-linked glycans from the mAb molecule is essential before thisanalysis. Otherwise, the size-based elution order on the HILIC columnwill be complicated by the presence or absence of glycans and thedifferent glycoforms.

Example 2 Free Light Chain Results

Multiple light chain-related impurities were detected in HILIC-MSanalysis of the mAb-1 sample (FIGS. 2A-2E). The extracted ionchromatograms (XICs) of each species suggested that they also exhibiteddifferent retention times during HILIC separation (FIG. 2A).Interestingly, the cysteinylated light chain (+˜119 Da) was identifiedas the major form of all light chain species present in mAb-1 sample andit is the only species visible by UV (FIG. 2D). The cysteinylation mayoccur as a result of the thiol-disulfide exchange reaction between theinter-heavy and light chain disulfide bond and a free cysteine molecule,which can be found in cytoplasm. In addition, glutathionylated lightchain (+˜306 Da) was also identified, with a retention time slightlylater than the cysteinylated light chain (FIG. 2E). Similar to thecysteinylation process, free glutathione (GSH) molecule, which can alsobe found in cytoplasm, should be responsible for this modification.Another interesting LC variant with a delta mass of +˜90 Da frompredicted mass was also observed (FIG. 2C). Subsequent peptide mappinganalysis using recombinant Lys-C protease located this modification tolight chain C-terminal Cys residue. Based on accurate delta mass (+89.98Da) and isotopic distribution, it was speculated that this modificationwas as a result of the thiol-disulfide exchange reaction between theinter-heavy and light chain disulfide bond and a 2-mercaptoacetic acidmolecule. However, how this compound was introduced or generated duringmAb production is currently under investigation. Although invisible inthe UV trace, unmodified free light chain can also be detected by moresensitive mass spectrometry analysis, along with a group of light chainvariants corresponding to the sequential clipping of the C-terminalamino acid residues (FIG. 2C). The resulting amino acid ladder as shownin the deconvoluted mass spectrum (FIG. 2C) matched with the C-terminalsequence of light chain (Gly-Glu-Cys). The removal of C-terminal aminoacid residues from the light chain might be attributed to the presenceof some carboxyl peptidases in the cell culture (Dick L W, Jr., Qiu D,Mahon D, Adamo M, Cheng K C. Biotechnology and bioengineering,100:1132-43 (2008); Hu Z, et al. Biotechnology and bioengineering,113:2100-6 (2016). Finally, another light chain variant with a massdecrease of ˜34 Da was also identified (FIG. 2B), suggesting possiblecysteine to dehydroalanine conversion via β-elimination on the lightchain C-terminal Cys residue. This reaction has been well studiedpreviously and identified as a major pathway for subsequent truncationat the antibody hinge region during storage (Cohen S L, Price C, VlasakJ. Journal of the American Chemical Society, 129:6976-7(2007)). It isnoteworthy all those light chain related impurities are not likely to bedetected by SEC method under native conditions, as they can still bindto the heavy chain via a strong non-covalent interaction despite thebroken inter-heavy and light chain disulfide bond.

Example 3 Fab Fragment Results

It is well known that the upper hinge region of heavy chains in an IgG1molecule is susceptible to hydrolysis leading to the formation of twocomplementary LMW species, a Fab fragment and a Fab-truncated species(Cordoba A J, Shyong B J, Breen D, Harris R J. Journal of chromatographyB: Analytical technologies in the biomedical and life sciences,818:115-21 (2005). The HILIC-MS method detected both species in themAb-1 sample. Four major Fab fragments with different masses wereidentified (FIGS. 3A-3D) and the truncation sites were located bycomparing the measured masses with the predicted masses, based on thecDNA-derived amino acid sequence. The amino acid pattern indicated inthe deconvoluted mass spectrum matched with the heavy chain hinge regionsequence (Cys-Asp-Lys-Thr-His-Thr-Cys). It is worth noting that any Fabfragment should have been readily removed during the purificationprocess due to the lack of the Protein A binding site. Therefore, thepresence of Fab fragments in the final drug product indicated that thoseimpurities were introduced as degradation products during samplestorage. The successful detection of those species emphasizes that theHILIC-MS method would be useful in stability and forced degradationstudies during mAb drug development.

Example 4 Half Antibody Results

Half antibody is formed as a result of inter-heavy and heavy chaindisulfide bonds at the hinge region scrambling into intra-heavy chaindisulfide bonds. Under native conditions, the four-chain structure of amAb molecule remains undisrupted by this scrambling, owing to the strongnon-covalent interaction between the two heavy chain C-terminal regions.However, when a method with denaturing conditions is used, such asSDS-PAGE or CE-SDS, the two half antibody molecules will dissociate fromeach other and appear as LMW impurities. During the HILIC-MS analysis ofmAb-1 product, the half antibody molecule was confidently identified(FIG. 3B). The good agreement between the measured mass (73,099.5 Da)and the predicted mass (73,100.1 Da) suggested that, unlike extensivelymodified free light chain, no substantial modification was associatedwith the formation of the half antibody, consistent with the disulfidebond scrambling pathway.

Example 5 Fab Truncates Species Results

As the complementary fragments to the Fab fragments after heavy chainhinge region truncation, Fab-truncated species were also identified inmAb-1 sample by the HILIC-MS method (FIG. 3C). On the HILIC column,those species were eluted right before the main peak as a partiallyresolved shouldering peak. The subsequent mass measurement confirmed theidentity of the Fab-truncated species and truncation sites were locatedat the hinge region consistent with those observed in the Fab fragments.Despite the inferior resolution in separation compared toelectrophoretic methods, the HILIC-MS method prevails in high fidelityand specificity by accurate mass measurement, making unambiguousidentification possible. Unlike the Fab fragments, the Fab-truncatedspecies are very difficult to remove from the main species duringpurification, due to the preserved Fc region that binds to Protein A.Monoclonal antibody molecules missing a Fab arm are expected to exhibitcompromised potency as one of the two target binding sites is notpresent. For bispecific mAb molecules, losing one Fab arm is detrimentalfor drug activity, since both Fab arms are essential to achieve thedesigned therapeutic functions. Therefore, the ability to detect andidentify those species directly in a drug substance sample is highlyimportant because time and resources are not only saved, but also avoidsthe introduction of possible artifacts during the enrichment process.

Example 6 H2L Species Results

The H2L species, comprised of two heavy chains and one light chain, arefrequently observed as the most abundant LMW impurities by SDS-PAGE andCE-SDS methods, but their identification is usually only supported byindirect and insufficient evidence. For example, N-terminal sequencinganalysis of the H2L-containing minor band on SDS-PAGE can reveal thefirst several amino acid residues of both heavy chain and light chain,and the signal intensity ratio between heavy chain and light chain mightsuggest a 2 to 1 ratio. In-gel digestion of the H2L-containing minorband followed by LC-MS based peptide mapping analysis can confidentlyconfirm the presence of both heavy chain and light chain. However, it isdifficult to establish an accurate ratio between heavy chain and lightchain. Both methods provide partial identification of H2L species, butneither can reveal a complete structure. In contrast, HILIC-MS methodoffers direct identification of H2L species at the intact protein level.In mAb-1 product, a homogeneous H2L species was observed to co-elutewith the Fab-truncated species slightly ahead of the intact antibody.The detected mass (122,851.0 Da) of the H2L species was approximately 34Da smaller than the predicted molecular weight of a H2L molecule(122,884.5 Da). The β-elimination of a heavy chain cysteine residue,presumably the one originally forming the inter-heavy and light chaindisulfide bond, was the root-cause of forming H2L species in mAb-1sample. This level of confidence and detailed information cannot beachieved by other well-established purity assays, making this methodvery valuable in providing essential identification for those LMWimpurities. It is believed that this study is the first case where H2Lspecies is shown to be directly detected by intact mass analysis in amAb drug substance sample without prior enrichment (FIG. 3D).

Example 7 LMW Impurities Generated Under Forced Conditions by LimitedReduction Results

Comparing to the HILIC-MS analysis of the mAb-1 sample, SDS-PAGE andCE-SDS methods frequently identified more LMW impurities, including theheavy chain dimer and free heavy chain. In order to demonstrate thecapability of the new HILIC method in detecting those impurities, thedeglycosylated mAb-1 sample was further treated with reductant(dithiothreitol (DTT) and L-cysteine) under native conditions tofacilitate the formation of various LMW impurities. It has been reportedthat under native conditions, only inter-chain disulfide bonds in a mAbmolecule can be reduced by DTT, since they are the onlysolvent-accessible disulfide bonds in the molecule (Liu H, et al.Analytical chemistry, 82:5219-26 (2010)). After briefly exposing mAb-1molecule to DTT or L-cysteine, LMW impurities with different heavy chainand light chain combinations can be generated, including free lightchain, free heavy chain, half antibody, heavy chain dimer and H2Lspecies. As shown in FIG. 4, these predicted LMW species were detectedin the HILIC separation with the elution order consistent with theirrelative size. In addition to those observed in the untreated mAb-1sample, two more LMW species, specifically, the free heavy chain speciesand the heavy chain dimer species, were observed to elute between thefree light chain and half antibody, and slightly after the half antibody(partially resolved peak), respectively.

In addition to elution order-guided assignment, the subsequent massspectrometry analysis following HILIC separation further confirmed theidentity of each impurity based on accurate mass (FIGS. 5A-5F).Interestingly, the light chain species in the DTT-treated mAb-1 sampleexhibited a very different retention time than the light chain speciesobserved in the untreated sample (FIG. 4). Based on the accurate massmeasurement, the difference in retention time was attributed to thecysteinylation on light chain that was present in the untreated samplebut absent in the DTT-treated sample. Consistently, when mAb-1 wastreated with L-cysteine, a small fraction of the light chain species wasobserved at the same retention time as the light chain species in theuntreated sample, suggesting the occurrence of cysteinylated light chainin the L-cysteine-treated sample. Finally, the measured mass of H2Lspecies in DTT-treated sample (122,884.5 Da) was in agreement with thepredicted mass of the unmodified H2L species (122,884.5 Da) because itis predominantly composed of the H2L species generated via disulfidebond reduction (predicted mass: 122,884.5 Da) and low levels ofpre-existing H2L species generated via β-elimination (predicted mass:122,850.4 Da)

While in the foregoing specification this invention has been describedin relation to certain embodiments thereof, and many details have beenput forth for the purpose of illustration, it will be apparent to thoseskilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention.

All publications mentioned throughout this disclosure are incorporatedherein by reference in their entirety.

All references cited herein are incorporated by reference in theirentirety. The present invention may be embodied in other specific formswithout departing from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

We claim:
 1. A protein drug product comprising: a protein drug and anexcipient, wherein the protein drug product comprises between 0.05% and30.0% (w/w) of low molecular weight protein drug impurities.
 2. Theprotein drug product of claim 1, wherein the protein drug product isselected from the group consisting of an antibody, a fusion protein,recombinant protein, or a combination thereof.
 3. The protein drugproduct of claim 1, wherein the low molecular weight protein drugimpurities are selected from the group consisting of degradationproducts, free light chain, half antibody, H2L, H2, HL, HC, or acombination thereof.
 4. The protein drug product of claim 1, wherein thedrug product comprises between 0.05% to 25% w/w of low molecular weightprotein drug impurities.
 5. The protein drug product of claim 1, whereinthe drug product comprises between 0.05% to 15% w/w of low molecularweight protein drug impurities.
 6. The protein drug product of claim 1,wherein the drug product comprises between 0.05% to 10% w/w of lowmolecular weight protein drug impurities.
 7. The protein drug product ofclaim 1, wherein the drug product comprises between 0.05% to 5% w/w oflow molecular weight protein drug impurities.
 8. A method forcharacterizing low molecular weight protein drug product impuritiescomprising: i) deglycosylating a protein drug product sample; ii)separating protein components of the protein drug product sample byhydrophilic interaction chromatography; iii) analyzing the separatedprotein components by mass spectroscopy to characterize low molecularweight protein drug product impurities in the protein drug productsample, wherein the protein drug product is as defined in claim
 1. 9.The method of claim 8, wherein the protein drug product sample is from afed-batch culture.
 10. The method of claim 8, wherein the protein drugproduct is selected from the group consisting of an antibody, a fusionprotein, recombinant protein, or a combination thereof.
 11. The methodof claim 8, wherein the low molecular weight protein drug productimpurity is characterized as a low molecular weight protein drug productimpurity selected from the group consisting of free light chain, halfantibody, H2L, H2, HL, HC, or a combination thereof.
 12. A method ofproducing an antibody, comprising: i) culturing cells producing theantibody in a cell culture; ii) obtaining a sample from the cellculture; iii) characterizing and quantifying low molecular weightimpurities in the sample according to the method of claim 8, and iv)modifying one or more culture conditions of the cell culture to reducethe amount of characterized low molecular protein drug impuritiesproduced during cell culture of the antibody.
 13. The method of claim12, wherein the one or more conditions of the cell culture that arechanged to reduce the amount of low molecular weight protein drugimpurities are selected from the group consisting of pH, cell density,amino acid concentration, osmolality, growth factor concentration,agitation, gas partial pressure, surfactants, or combinations thereof.14. The method of claim 12, wherein the cells are selected from thegroup consisting of bacterial cells, yeast cells, Chinese Hamster Ovary(CHO) cells (e.g. CHO K1, DXB-11 CHO, Veggie-CHO), COS cells (e.g.COS-7), retinal cells, Vero cells, CV1 cells, kidney cells (e.g. HEK293,293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa cells, HepG2 cells, WI38cells, MRC 5 cells, Colo25 cells, HB 8065 cells, HL-60 cells, lymphocytecells, e.g. autologous T cells, Jurkat (T lymphocytes) or Daudi (Blymphocytes), A431 (epidermal) cells, U937 cells, 3T3 cells, L cells,C127 cells, SP2/0 cells, NS-0 cells, MMT cells, stem cells, tumor cells,and a cell line derived from any of the aforementioned cells.
 15. Themethod of claim 12, wherein the cells are hybridoma cells or quadromacells.
 16. An antibody produced by the method of claim
 12. 17. Theantibody of claim 16, comprising 0.05 and 30.0% (w/w) of low molecularweight protein drug impurities.
 18. A system for characterizing lowmolecular weight drug impurities, comprising: a hydrophilic interactionliquid chromatography system comprising a hydrophilic interaction liquidchromatography (HILIC) column linked to at least two mobile phasecolumns, wherein the HILIC column is in fluid communication with a massspectroscopy system.
 19. A method for determining the stability andforced degradation studies of a protein drug product using the system ofclaim 18.